The site and mechanism of action of epidermal growth factor (EGF) on acid secretion by rat isolated parietal cells were investigated by using the intracellular accumulation of the weak base aminopyrine as an index of secretory activity. When parietal cells were stimulated with histamine (0.5 mM), the concentration of EGF required for half-maximal inhibition of acid secretion was 19 nM, with a maximally effective concentration of EGF producing 38% inhibition of secretory activity. EGF did not inhibit secretion stimulated by 0.1 mM-carbachol, or by 30 microM-, 56 microM-, 100 microM- or 1000 microM-dibutyryl cyclic AMP, low concentrations of which produced a secretory response comparable with that obtained with 0.5 mM-histamine. Addition of 0.1 mM-3-isobutyl-1-methylxanthine (IBMX) substantially increased aminopyrine accumulation in the presence of 0.5 mM-histamine. The inhibitory action of EGF on histamine-stimulated secretion was blocked by 0.1 mM-IBMX, even if low concentrations of histamine were used to generate aminopyrine accumulation ratios similar to those obtained with 0.5 mM-histamine alone. The cyclo-oxygenase inhibitor flurbiprofen (1-100 microM) and the cyclo-oxygenase and lipoxygenase inhibitor nordihydroguaiaretic acid (10-100 microM) did not affect the inhibitory action of EGF. The pattern of inhibition of secretion produced by the activator of Ca2+-sensitive phospholipid-dependent protein kinase, 12-O-tetradecanoylphorbol 13-acetate, was markedly different from that produced by EGF. In conclusion, a major site of the action of EGF on acid secretion in the intact stomach is probably a decrease in the stimulatory effect of histamine by a mechanism which does not involve Ca2+-sensitive phospholipid-dependent protein kinase or the production of prostaglandins, but which might involve enhancement of cyclic AMP phosphodiesterase activity.

Sites at which the calcium-sensitive phospholipid-dependent protein kinase, protein kinase C, may influence acid secretion have been investigated in rat isolated parietal cells. In both crude and enriched preparations of parietal cells incubated in a medium containing 100 mM K+, the activators of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA) and 1-oleoyl-2-acetyl-glycerol (OAG), produced a dose-dependent stimulation [half-maximally effective concentration (EC50) values of 1 nM and 70 microM, respectively] of aminopyrine accumulation, an index of the sequestration of acid in the cell. In a medium containing 4.5 mM K+, and with no added secretagogues, TPA and OAG did not affect aminopyrine accumulation. Histamine-stimulated aminopyrine accumulation was inhibited by TPA [half-maximally effective inhibitory concentration (IC50) of 2.9 nM]. TPA reduced the histamine-stimulation of the adenosine 3',5'-cyclic monophosphate (cAMP) content of parietal cells (47% inhibition at 100 nM TPA) but also inhibited aminopyrine accumulation at or distal to cAMP-dependent protein kinase. Activators of protein kinase C can produce multiple effects on secretory activity in the rat parietal cell.

Histamine (0.5 mM) stimulated the cyclic AMP content of cell suspensions containing > 80 parietal cells. Epidermal growth factor (EGF) inhibited this stimulatory effect of histamine, but had no effect on basal cyclic AMP content. The half-maximally effective concentration of EGF for inhibition of histaminestimulated cyclic AMP was 3.9 nm. The equivalent measurement for the inhibition of histamine-stimulated aminopyrine accumulation was 3.0 nM. Aminopyrine accumulation was measured because it provides an index of the secretory activity of the cell. The cyclic AMP phosphodiesterase inhibitor 3-isobutyl-Imethylxanthine (IBMX) prevented the inhibitory effect of EGF on cyclic AMP content. This effect of IBMX was not caused by its ability to raise cellular cyclic AMP content in the presence of histamine. Prevention by IBMX of the inhibitory action of EGF on histamine-stimulated aminopyrine accumulation had been shown previously [Shaw, Hatt, Anderson & Hanson (1987) Biochem. J. 244, 699-704]. EGF stimulated prostaglandin E2 (PGE2) production in the cell fraction containing > 800% parietal cells, with the halfmaximally effective concentration being 7.5 nM. EGF was ineffective in stimulating PGE2 production if the cell fraction was depleted of parietal cells (12 %), or if 0.5 mM-histamine was added to the enriched parietalcell fraction. In conclusion, EGF may inhibit histamine-stimulated acid secretion by decreasing the cyclic AMP content of parietal cells. This effect could be mediated by an increase in cyclic AMP phosphodiesterase activity, but it is unlikely to involve an effect of EGF on parietal-cell prostaglandin production. INTRODUCTIONEpidermal growth factor (EGF) is a polypeptide which was first isolated from the mouse submaxillary gland. In the rat, intraperitoneal or intragastric administration of EGF causes a trophic response in the gastroduodenal mucosa (Dembinski et al., 1982). Intravenous or subcutaneous EGF inhibits gastric acid secretion (Bower et al., 1975;Dembinski et al., 1982;Gregory, 1985), but intragastric administration of EGF only inhibits acid secretion when very high doses are used (Konturek et al., 1981). Thus EGF in the gastric lumen could be derived from saliva and might play a role in day-to-day maintenance of the gastric epithelium (Skinner et al., 1984), but luminal EGF is unlikely to regulate gastric acid secretion. Inhibition of acid secretion by EGF might occur during acute damage to the epithelium, and the absence of acid might facilitate the process of repair (Gregory, 1985;Shaw et al., 1987). In such circumstances EGF could be derived from platelets (Shaw et al., 1987 J. F. Hatt and P. J. Hanson about 20 % parietal cells, was resuspended in Eagle's minimum essential medium containing 36 ml of isoosmotic Percoll/100 ml, 12 mM-Hepes, 0.6 mg of bovine serum albumin/ml, 0.3 mM-dithiothreitol and 1.9 mm-EGTA, pH 7.35. This mixture was centrifuged for 13 min at 30000 gayv at 4°C with a 200 angle rotor in a MSE Superspeed 50 centrifuge. The cell fraction removed from the top 20 % of the gr...

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Ihisioti o/'Hio/o&y, l ' i i i r r t~i i i~~~~r i t i~~i r lScicticvs Iti.siitirtc, A.stoti Utiitwsity, Astori 7i.iirtigIc. Hirt)iitigiiirtti 114 7E7; U. K .Epidermal growth factor (EGF ) from mouse submaxillary glands is ;I polypeptide o f hi, 00.15 which stimulates cpithelial cell proliferation ;ind which inhibits gastric acid secretion both iti iii.o I I I a n d iri ~i i r o 12. 3 I . Inhibition of pepsinogen secretion by k